Kiwifruit Enzyme: a New Plant Coagulant for the Development of Cottage Cheese

International Journal of Food Science and Nutrition

International Journal of Food Science and Nutrition ISSN: 2455-4898 Impact Factor: RJIF 5.14 www.foodsciencejournal.com Volume 3; Issue 1; January 2018; Page No. 12-19

Kiwifruit enzyme: A new plant coagulant for the development of cottage cheese

* Swati Sharma, Devina Vaidya Department of Food Science and Technology, YSP University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh, India

Abstract In the present work, extraction of kiwifruit enzyme, partial purification, characterization, optimization of enzyme concentration, quality of cottage cheese prepared and their comparative study with animal rennet was studied. In the experiment, first enzyme was extracted at various stages of maturity followed by partial purification, characterization and optimization of enzyme concentration was done. Results showed that kiwifruit enzyme has maximum enzyme activity at immature stage and partial purification with ammonium sulphate precipitation method at 40-60 per cent shows the highest yield. Characterization of enzyme through Response Surface Methodology (RSM) shows that enzyme was stable at pH (8) and temperature (450C). The quality of cottage cheese prepared with kiwifruit enzyme was comparable to the rennet cottage cheese. Therefore, it was finally concluded that animal origin rennet can be completely or effectively replaced by employing plant kiwifruit partially purified enzyme at the rate of 0.5 per cent for cottage cheese production.

Keywords: kiwifruit enzyme, characterization, partial purification, cottage cheese, rennet

1. Introduction 2. Materials and methods Kiwifruit (Actinidia deliciosa) is native to Southern China [10]. 2.1 Procurement of raw material Kiwifruit is very popular in human diet due to its pleasant The raw material i.e. kiwifruit (Actinidia deliciosa) was taste and high content of vitamin C, minerals (potassium, procured from Kiwifruit Orchard, Department of Fruit phosphorus, iron) and low calorific value. Kiwifruits are good Science, milk from Dairy Farm, Department of Silviculture sources of folate, potassium and contain large amounts of and Agroforestry, YSP University of Horticulture and vitamin E in the seeds. Moreover, kiwifruit juice is known to Forestry, Nauni-Solan (India), while rennet was procured from contain highly active proteolytic enzymes [14]. Enzymes are Guru Angad Dev Veteniary and Animal Sciences University, widely used as technological aids in several food processes. Ludhiana, Punjab (India). However other materials used for Proteolytic enzymes are multifunctional class of enzymes [22]. producing cottage cheese viz. starter culture and salt were Henceforth kiwifruit enzyme as a proteolytic enzyme can be obtained from the local market of Nauni-Solan, Himachal used in food industry as milk clotting enzyme. Kiwifruit Pradesh (India). enzyme has the ability to form milk clots so that the enzyme is fully compatible with conditions used in cheese manufacture 2.2 Estimation of soluble protein in kiwifruit (optimum activity at 40-42oC, mildly acidic pH values). Protein concentration in kiwifruit was determined using Analysis of products produced by hydrolysis by using procedure followed by Lowry [16]. actinidin showed that the preferred substrate for this enzyme is β-casein, followed by k-casein, which is hydrolysed into a 2.3 Extraction of crude enzyme small number of larger peptides [18]. Milk coagulation is a The procedure followed by Thimmaiah [31] was employed for basic step in cheese manufacturing and is most commonly extraction of crude enzyme from kiwifruit. First the kiwifruit achieved by addition of chymosin (rennet), milk clotting tissues were homogenized in pestle-mortar with phosphate enzyme preparation obtained from the stomach contents of the buffer (pH 8.0) under cool condition (0-50C) and extract was unweaned calf [19]. The worldwide increase in cheese centrifuged at 10,000 rpm for 10 min in refrigerated production, alongside with the reduced supply of calf rennet centrifuge. The supernatant was labeled as “Crude enzyme”. and higher prices, have led to an increase in the demand for alternative sources of milk coagulants [6]. In addition, the use 2.4 Partial purification of enzyme of animal rennet has consumer constraints due to religious The procedure followed by Sadasivam and Manickam [28] was reasons (e.g. Judaism and Islam), diet (vegetarianism), or bans employed for partial purification of enzyme extracted from on genetically engineered food [27]. For these reasons, enzymes kiwifruit. First partial purification of enzyme was done by extracted from plants have become a subject of growing using ammonium sulphate fractionation method as mentioned interest in dairy technology. Henceforth, present study was above. The crude extract of kiwifruit was precipitated by planned to assess the effect of kiwifruit enzyme on quality ammonium sulphate using different concentrations (0-90 per attributes of cottage cheese as potential vegetable coagulant to cent). Precipitation was carried out at 0-50C and the replace the animal origin rennet. precipitate was recovered by centrifugation. The supernatant

12 International Journal of Food Science and Nutrition was discarded and the sediment from each concentration was version 7.0 (Stat Ease, Inc, Minneapolis, USA). The details of dissolved in phosphate buffer solution (pH 8.0) and dialyzed experimental design plan for characterization of enzyme are over night against the same buffer. The dialyzed enzyme was given in Table 2. used for further studies. Observations recorded dring partial purification are- Table 1: Range of values for the RSM

Variables -1 0 +1 [4] 2.4.1 Specific activity pH 4 8 12 Specific activity was calculated by dividing the total enzyme Temperature (0C) 30 45 60 activity with total protein. Incubation Time (min) 10 20 30

2.5.1 Experimental design and equation modelling Second-order experimental design, i.e. Central Composite Design (CCD) with three factors at three levels was employed 2.4.2 Fold purification [4] to investigate the first and higher-order main effects of each Fold purification was calculated by dividing the specific factor as well as interactions among them. The design activity of partially purified enzyme with specific activity of involved eight center design points with ‘α’ value being ± 2. crude enzyme. The three coded levels investigated in the current study were - 1, 0, and 1 as shown in table. The enzyme assay conditions were optimized on the basis of highest enzyme activity. The experimental results obtained by the characterization of

[4] enzyme were applied to obtain the regression models. Based 2.4.3 Per cent yield / Recovery of enzyme on experimental design (Table 1), the cubic model could be Percent yield / Recovery was calculated by dividing the total fitted in case the lacks of fit of the quadratic or linear models enzyme activity of partially purified enzyme with total activity were statistically significant (p<0.05), Where Y , evaluated of crude enzyme. i response; b terms were coefficients estimated by the least square

2.5 Characterization of enzyme Response Surface Methodology (RSM) was used for the characterization of the enzyme by keeping the effects of pH, method; x terms were dependent variables [9]. Quality of the temperature, time of incubation as independent variables and models fitness was evaluated by ANOVA, in which the enzyme activity as a dependent variable. As per the design repetition supplied the freedom degree to obtain the pure treatments chosen were shown in Table 1. The different error. Calculations and graphics were performed by opstat. combinations were made as per the expert RSM design This was used at the beginning of this study.

Table 2: Experimental plan of characterization of enzyme as per the design

Treatments Factor 1 A:pH Factor 2 B:Temp (0C) Factor 3 C: Time of incubation (min) T1 8.00 45.00 20.00 T2 4.00 30.00 10.00 T3 1.27 45.00 20.00 T4 8.00 45.00 20.00 T5 8.00 45.00 20.00 T6 12.00 60.00 30.00 T7 8.00 45.00 20.00 T8 8.00 45.00 36.82 T9 12.00 30.00 10.00 T10 8.00 70.23 20.00 T11 4.00 30.00 30.00 T12 12.00 60.00 10.00 T13 8.00 45.00 20.00 T14 4.00 60.00 30.00 T15 4.00 60.00 10.00 T16 14.73 45.00 20.00 T17 8.00 45.00 20.00 T18 12.00 30.00 30.00 T19 8.00 19.77 20.00 T20 8.00 45.00 3.18

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2.6 Enzyme assay 2.9.2 Proximate analysis The procedure followed by Thimmaiah [31] was employed for The moisture content, crude protein, crude fat and ash content assessment of enzyme assay with slight modifications. For were determined in triplicates following methods of AOAC [2]. this, one ml of reaction mixture containing 1 per cent casein in 1 ml of 0.05 M phosphate buffer, pH 8.0 and 1 ml of enzyme 2.9.3 Calcium content was incubated at 45°C for 20 min. After 2hr, the reaction was The AOAC method [2] was followed for estimation of calcium stopped by adding 2ml of cold 10 per cent trichloroacetic acid content. For that one gram of cheese sample digested in 20 ml (TCA) and for blank, immediately after incubation reaction concentrated sulphuric acid till 3-4 ml extract was remained was stopped with 10 per cent TCA. After 2 hour, the culture after that it was diluted with distilled water to make 100 ml filtrate was centrifuged at 10,000 rpm for 10 min in final volume. Then calcium content was estimated with flame refrigerated high speed research centrifuge (Model TC 4100 photometer with standard procedure. F/RC 4100 F) to remove the precipitate and absorbance of the supernatant was read spectrophotometrically at 660 nm. 2.9.4 Total carbohydrates Enzyme activity was calculated by measuring mg of tyrosine The total carbohydrate content as per cent dry weight basis equivalent released and compared with the standard. One unit was determined mathematically. (U) of enzyme activity represents the amount of enzyme Total carbohydrate (%) = 100 - (% crude protein + % required to liberate 1 μg of tyrosine under standard assay crude fat + % moisture content + % ash content) conditions.

2.9.5 Energy value 2.7 Optimization of enzyme concentration The energy value of samples was calculated using the Enzyme concentration was standardized on the basis of milk [3] following relation. clotting activity with a slight modification . The substrate (skimmed milk) was prepared and the pH was adjusted to 6.5. Energy value in (Kcal) = (Crude protein x 4.1) + (Crude The substrate (10 ml) was pre-incubated for 5 min at 370C and fat x 9.3) + (Carbohydrate x 4.1) different concentration (0, 2, 4, 6, 8, and 10 %) of enzyme extract was added and the curd formation was observed at 2.10 Texture analysis 370C while manually rotating the test tube from time to time. The texture profile analysis of cheese samples were measured The end point was recorded when discrete particles were by using Texture Analyzer, TAXT2i (Stable 70 Microsystems, discernible. One milk clotting unit is defined as the amount of UK), equipped with a cell charge of 25 kg. The texture profile enzyme that clots 10 ml of the substrate within 40 min. was obtained by a double compression test of the cheese samples, at room temperature, using a cylindrical compression MCA (U/ml) = (2400/clotting time (sec)) x Dilution factor probe with a 75 mm diameter (P/75). Test parameters included hardness, fracturability, adhesiveness, springiness, Where MCA= milk clotting activity gumminess, chewiness, cohesiveness and resilience (Annexure IV). The instrument was operated at pre-test speed 2.8 Preparation of cottage cheese = 1.00 mm/s, test speed = 5.00 mm/s, post test speed = 5.00 Cottage cheese was manufactured with some modifications [12, mm/s, strain rate = 50%, trigger force = 0.04903 gm, force 29]. About 2000 ml of pasteurized milk was heated in a pan and data acquisition rate of 100 pps. while stirring on the induction heater. Yogurt as starter culture (4-5 %) was added into pan when the temperature of 45oC 2.11 Sensory attributes of cottage cheese reached after which kiwifruit enzyme was immediately added The products were subjected to organoleptic analysis as per to the milk using a sterile pipette and allowed for setting in the procedure followed by Larmond, [15]. In the study, sample incubator at 21oC for 14 hrs. After the milk was fully of coded cheese was served in cleaned white plate to panelist coagulated and settled, cutting of cougulam into smaller of all age groups of both sexes at room temperature (250C) for pieces was done. The process of cooking (1 hr) and then sensory evaluation by using Hedonic scale (annexure ) where pressing out the remaining whey, tied and pressed with a 1 = Dislike extremely and 9 = Like extremely. known weight to dry to a constant weight (approximately after 2hrs) was performed. For preservation, salt (at the rate of 1% 2.12 Statistical analysis of curd) was also added into cheese. The cheese was then The data pertaining to the sensory evaluation of cottage cheese removed into a sterile container and stored in the fridge at 4oC were analyzed according to Randomized Block Design overnight or analyzed immediately. Similar procedure of (RBD), while the data on chemical characteristics of product cheese making with rennet was followed in place of kiwifruit was analyzed statistically by following Completely enzyme treated as control. Randomized Design (CRD) [8].

2.9 Effect of kiwifruit enzyme on physico-chemical 3. Results and Discussion attributes of cottage cheese The aim of the present study was to assess the effect of 2.9.1 Yield of cheese and whey enzymes extracted from the kiwifruit on milk clotting and The fresh cottage cheese was studied for their quality quality attributes of cottage cheese prepared. In this study, characteristics i.e. Cheese yield and whey yield was calculated first kiwifruit enzyme was extracted at various stages of fruit by the standard methods of Mahajan and Chaudhari [20]. maturity followed by their partial purification and

14 International Journal of Food Science and Nutrition characterization. Partially purified kiwifruit enzyme was Table 3: Protein content and enzyme activity of kiwifruit employed for production of cottage cheese. Finally the quality (Mean±SE) attributes of cottage cheese produced with kiwifruit enzymes Protein Enzyme Stage were compared with rennet cheese. (mg/gm) activity (µg/gm) Immature (TSS<6.5 0 B) 0.42 ± 0.20 200.32 ± 0.20 3.1 Extraction of kiwifruit enzyme at various stages of Mature (TSS=6.5 0 B) 0.28 ± 0.10 131.50 ± 0.20 fruit maturity Ripened (TSS<14 0 B) 0.25 ± 0.20 130.25 ± 0.20 This was done in order to estimate the right stage of enzyme extraction. Three different stages of fruit maturity i.e. 3.2 Partial purification of kiwifruit enzyme immature stage (7 days before attaining the commercial In the Table 4, purification profile of kiwifruit enzyme was harvest date (TSS<6.50B)), mature (commercial harvest date presented. With ammonium sulphate precipitation, the (TSS=6.50B)) and ripened stage (8-10 days after commercial fractionation was carried out in different ranges i.e. 20-90 per harvest date (TSS<140B)) was taken. The data in Table 3 cent. Each fraction was assayed for its protease activity. represents the protein content and enzyme activity of kiwifruit Highest enzyme activity, yield and purification fold was found at various stages of fruit maturity as protein content indirectly with 40-60 per cent concentration of ammonium sulphate, i.e. represents the enzyme. The highest protein content and 86 per cent protease enzyme yield of 1.65 purification fold enzyme activity (0.42±0.20 mg/gm and 200.32±0.20 µg/gm) and 0.86 /mg of protein specific activity were found in was observed at immature stage of fruit followed by mature kiwifruit. However Chaiwut et al. [7] and Otani et al. [26] (0.28±0.10 mg/gm and 131.50±0.20 µg/gm) and then ripened reported the highest activity of protease enzyme extracted (0.25±0.20 mg/gm and 130.25±0.20 µg/gm) stage from fig, precipitated with 50-70 per cent ammonium respectively. It is shown from the experiment that in kiwifruit sulphate. Based on results, it is concluded that kiwifruit maximum enzyme activity was shown at immature stage. enzyme was precipitated in two steps, in which it was possible Similar findings were reported by Whitaker, [32] that the to recover more than 86 per cent of its initial activity with the enzyme activity of Ficus carica fruit was highest when they purification factor of 1.65. were unripened green.

Table 4: Purification profile of kiwifruit enzyme

Observations Protein Enzyme activity Specific activity Purification % Purification step (mg/gm) (µg/gm) (/mg protein) fold Yield Crude enzyme 0.42 220.00 0.52 1 100 Ammonium sulphate precipitation (40-60%) 0.22 190.00 0.86 1.65 86

3.3 Characterization of kiwifruit enzyme statistical model can explain 58.82% of variability in the The results obtained from response surface design for enzyme response. Adequate precision measures signal to noise ratio. activity are presented in figures 1(a-c). These figures depict For enzyme activity, an adequate precision of 4.815 was the expected response of enzyme activity and correlation recorded (A ratio greater than 4 is desirable), which indicates between the independent variables in three dimensional plots. an adequate signal. Figure (a) shows pH at X- axis and time of incubation at Y- The analysis of both pH and temperature dependence and axis whereas figure (b) represents pH at X- axis and stability of kiwifruit enzyme (Figures 1 a, b, c) suggests that temperature at Y- axis and in figure (c) temperature at X- axis the enzyme might be fully compatible with conditions used in and time of incubation at Y- axis. From figure it was clearly cheese manufacture as well as with rennet action. shown that with increase in pH (8.0), temperature (450C) and Furthermore, the decrease in the enzyme activity observed time of incubation (20 min) up to a certain limit significant after 20 min of incubation and that ensures a lowering of the increase in enzyme activity was observed up to a certain level. proteolysis rate during the cheese making process, thus After that there was decrease in enzyme activity with further limiting the amount of short peptides that might be responsible increase in value of independent factors. Maximum enzyme for the enhancement of bitter taste. activity of 342.00 µg/gm was observed at 8.0 pH and at 450C However, Hullikere et al. [13]observed the protease enzyme of temperature. The regression equation coefficients were papaya with maximum activity at 400 C temperature and 7 pH, calculated and the data were fitted to a second-order whereas Omar et al. [25] characterized the proteolytic enzyme polynomial equation. So that the response (enzyme activity) of pineapple and found optimum temperature and pH 650 C could be expressed in terms of the following regression and 7.5 respectively and further reported that bromelain equation: retained more than 90 per cent of its original activity after a period of 1 hr incubation at 550C. Enzyme activity = 340.20 + 2.36A + 19.89B + 76.19C - 96.15A2 - 1.15 B2 + 13.88C2 - 24.25AB - 9.58AC + 6.08BC 3.4 Preparation of cottage cheese The preparation of cottage cheese required the optimization of Analysis of variance gave the enzyme activity as a function of enzyme concentration. The enzyme concentration was the initial values of parameters. The coefficient of optimized on the basis of milk clotting activity. determination (R²) was calculated as 0.5882 indicating that the Fig 1(b) Effect of pH and temperature 15 on enzyme activity

International Journal of Food Science and Nutrition

Fig 1(a): Effect of pH and time of Fig 1(b): Effect of pH and temperature on Fig 1(c): Effect of temperature and time of incubation on enzyme activity enzyme activity incubation on enzyme

3.4.1 Milk clotting activity clotting time) were found best for cottage cheese production Perusal of data in Table 5 indicates the optimized as indicated by first appearance of solid material in the milk. concentration of kiwifruit enzyme and rennet on the basis of However milk-clotting activity estimated by Cavalcanti et al. milk clotting activity for the preparation of cottage cheese. [6] in different precipitation fractions of the Nocardiopsis sp Kiwifruit enzyme shows milk-clotting activity, which is were in the range of 1.14-20.00 U/ml whereas others reported correlated with enzyme concentration. The enzyme the clotting time of proteinase isolated from mature flowers, concentrations i.e. 0.5 per cent partially purified kiwifruit immature flowers and leaves of artichoke were 8, 90, 180 min enzyme (with milk clotting activity 1.45 U/ml and 30 min respectively [17,1].

Table 5: Optimized concentration of kiwifruit enzyme and rennet for the development of cottage cheese

Enzyme source Concentration (%) Milk clotting activity (U/ml) Clotting time (min) Rennet 1.00 3.81±0.1 10 Kiwifruit partially purified enzyme 0.50 1.45±0.1 30

3.5 Effect of kiwifruit enzyme on physico-chemical quality preparation of cheese. attributes of cottage cheese In one of the related work Mahajan and Chaudhari [20] and 3.5.1 Yield and protein content of cottage cheese Mahami et al. [21] reported 18.19-24.42 per cent yield of Results of the influence of kiwifruit enzyme on yield and cottage cheese prepared with different concentrations (0.5-2.0 protein content of cottage cheese are summarized in Table 6. ml) of moringa seed extract. However whey yield 63.60 to The yield of kiwifruit cottage cheese was comparatively found 66.60 per cent was reported by Ojedapo et al. [23]. The protein lower as compared to rennet cheese i.e. 19.00 ± 0.30 and content of 15.52 and 17.80 was recorded in both kiwifruit and 23.16 ± 0.30 per cent whereas the whey yield was 73.91 ± rennet cottage cheese respectively whereas 12.14 to 15.71 0.30 and 78.00 ± 0.10 per cent respectively. Despite of lower percent protein content was recorded by Mahajan and yield as compared to rennet, cottage cheese prepared from the Chaudhari, [20] in cheeses prepared with plant latex of kiwifruit enzyme may be considered a promising alternative Euphorbiaceae family. of calf rennet for the coagulation of milk and/or for

Table 6: Effect of kiwifruit enzyme on yield and protein content of cottage cheese

Treatments % yield of cottage cheese Protein content in cheese (%) Whey yield (%) Rennet (1%) 23.16 ± 0.30 17.80 ± 0.30 73.91 ± 0.30 Partially purified kiwifruit enzyme (0.5%) 19.00 ± 0.30 15.52 ± 0.10 78.00 ± 0.10

3.5.2 Proximate analysis of cottage cheese observed in titratable acidity, ash and calcium content of The effect of kiwifruit enzyme on proximate analysis of kiwifruit cottage cheese. The results were parallel with the cottage cheese is presented in Table 7. The significant findings of Oladipo and Jadesimi [24].Total carbohydrates and increase in moisture content was found in cottage cheese energy value were found 7.27 to 13.72 per cent and 138.47 to prepared with kiwifruit enzyme. However fat content was 165.73 (Kcal) on calculation basis respectively, while Buriti et decreased in plant rennet cottage cheese as compared to al. [5]; Mahajan and Chaudhari [20] found calcium, carbohydrate animal rennet cottage cheese. The difference between two and energy value in the range of 1.38-12.29 g/kg, 6.14 to 6.84 types of cheese might be due to change in proteolysis rate. per cent and 272.41 to 318.80 Kcal/g respectively in cheese Our findings were closely associated with the findings of prepared with plant extracts. Mahami et al. [21] whereas non-significant changes were

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Table 7: Effect of kiwifruit enzyme on physicochemical characteristics of cottage cheese

Parameters Moisture Titratable Crude fat Ash Calcium Total Energy value Treatments (%) acidity (%) (%) (%) (%) Carbohydrates (%) (kcal/100g) Rennet (1%) 68.33 0.085 4.10 2.50 20.50 7.27 140.91 Kiwifruit partially purified enzyme (0.5%) 68.80 0.086 4.00 2.50 20.47 9.18 138.47 CD0.05 0.03 NS* 0.07 NS* NS* 0.17 0.67 *NS- non significant

3.6 Texture profile analysis (TPA) of cottage cheese highest cohesiveness (0.73) and springiness (0.86 mm) and the Figure 2(a-b) shows the effect of rennet and kiwifruit enzyme latter had the highest hardness (9600.85 N), fracturability on the TPA characteristics of cottage cheese. Regarding TPA (11910.19 N), Guminess (N), chewiness (N.mm) and lowest parameters both kiwifruit enzyme cottage cheese and rennet springiness (mm) and cohesiveness. Differences can be related cheese presented significant differences between each other. to differences in the degree of proteolysis during preparation. With the addition of kiwifruit enzyme, the cottage cheese had Similar behaviors in findings were reported by Sheibani et al. the lowest hardness (6668.65 N), fracturability (7349.70 N), [30] i.e. hardness and gumminess decreased from 13.17 to 11.06 guminess (4908.08 N), chewiness (4250.39 N.mm) and N and 10.00 to 7.06 N respectively in butter cheese.

Fig 2(a): Texture profile analysis (TPA) of cottage cheese with rennet

Fig 2(b): Texture profile analysis (TPA) of cottage cheese with kiwifruit enzyme (protease)

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3.7 Sensory characteristics of cottage cheese purified kiwifruit enzyme scored highest for overall Appraisal of data presented in fig 3 indicates the acceptability compared to cottage cheese prepared with sensory/organoleptic characteristics i.e. texture, colour, taste, rennet. flavor and overall acceptability of cottage cheese. Cottage The results were parallel with Galan et al. [11] reported that cheese was prepared as per the standardized recipe with cardoon (vegetable) rennet cheese exhibited softer texture and kiwifruit enzyme and compared with cheese prepared with higher creaminess scores as compared with the calf rennet commercial rennet (microbial source) for sensory cheeses. characteristics. The cottage cheese prepared with partially

Fig 3: Effect of kiwifruit enzyme on sensory characteristics* of cottage cheese *on 9-point hedonic scale

4. Conclusion York, 1970, 446-459. From this study it was concluded that kiwifruit enzyme is 4. Blaber SI, Ciric B, Christophi GP, Bernett MJ, Blaber M, potential vegetable source of coagulant for manufacturing of Rodriguez M et al. Targeting kallikrein 6 proteolysis cottage cheese without any adverse effect on taste and texture. attenuates CNS inflammatory disease. The Federation of The immature stage of fruits is superior for extraction of American Societies for Experimental Biology Journal. enzyme with maximum proteolytic activities for milk-clotting 2004; 18(7):920-922. and preparation of cottage cheese. Further, it is also show that 5. Buriti FC, Cardarelli HR, Filisetti TM, Saad SM. the enzyme is fully compatible with the physical–chemical Synbiotic potential of fresh cream cheese supplemented conditions utilized during cottage cheese manufacture (40–45 with inulin and Lactobacillus paracasei in co-culture with °C, sub-acid pH values). Henceforth, kiwifruit enzyme can be Streptococcus thermophilus. Food Chemistry. 2007; considered a promising alternative of natural calf rennet for 104(4):1605-1610. the coagulation of milk leading to new dairy products. 6. Cavalcanti MTH, Teixeira MFS, Lima-Filho JL, Porto ALF. Partial purification of new milk-clotting enzyme 5. Acknowledgements produced by Nocardiopsis sp. Bioresource Technology. This research had been conducted within the All India Co- 2010; 93:29-35. ordinated Research Project on “Post Harvest and Engineering 7. Chaiwut P, Nitsawang S, Shank L, Kanasawud P. Technology” at Nauni, Solan (HP), India. Comparative study on properties and proteolytic components of papaya peel and latex proteases. Chiang 6. References Mai Journal of Science. 2007; 34(1):109-118. 1. Abdalla MOM, Kheir SEO, El Owni OAO. Effect of 8. Cochran WG, Cox CN. Experimental Designs. New extraction method, ammonium sulphate concentration, Delhi: John Wiley and Sons, Inc. 1967. temperature and pH on milk-Clotting activity of Solanum 9. Dutta JR, Dutta PK, Banerjee R. Optimization of culture dubium fruit extract. Advance Journal of Food Science parameters for extracellular protease production from a and Technology. 2011; 3:40-44. newly isolated Pseudomonas sp. using response surface 2. AOAC. Association of Official Analytical Chemists. In and artificial neural network models. Process W. Hortwitz, (Ed.), Official Methods of Analysis. Biochemistry. 2004; 39:2193-2198. Academic Press, Washington, 1980, 1015. 10. Ferguson AR. Kiwi fruit a botanical review. Horticultural 3. Arima K, Yu J, Iwasaki S. Milk-clotting enzyme Review, Wesport: AVI Publishing. 1984, 1-64. from Mucorpusillus var. Lindt. In G. Perlmann, L Lorand 11. Galan E, Prados F, Pino A, Tejada L, Fernandez-Salguero (Eds.), Methods in Enzymology, Academic Press, New J. Influence of different amounts of vegetable coagulant

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